Diagnostic Performances of PET/CT Using Fibroblast Activation Protein Inhibitors in Patients with Primary and Metastatic Liver Tumors: A Comprehensive Literature Review

PET/CT using radiolabeled fibroblast activation protein inhibitors (FAPIs) is a promising diagnostic tool in oncology, especially when non-increased and/or physiologically high [18F]FDG uptake (as in liver parenchyma) is observed. We aimed to review the role of PET/CT using radiolabeled FAPIs in primary and/or metastatic liver lesions, and to compare their performances with more “conventional” radiopharmaceuticals. A search algorithm based on the terms “FAPI” AND (“hepatic” OR “liver”) was applied, with the last update on 1st January 2024. Out of 177 articles retrieved, 76 studies reporting on the diagnostic application of radiolabeled FAPI PET/CT in at least one patient harboring primary or metastatic liver lesion(s) were fully analyzed. Although there was some heterogeneity in clinical conditions and/or study methodology, PET/CT with radiolabeled FAPIs showed an excellent performance in common primary liver malignancies (hepatocarcinoma, intrahepatic cholangiocarcinoma) and liver metastases (mostly from the gastrointestinal tract and lungs). A higher tumor-to-background ratio for FAPIs than for [18F]FDG was found in primary and metastatic liver lesions, due to lower background activity. Despite limited clinical evidence, radiolabeled FAPIs may be used to assess the suitability and effectiveness of FAPI-derived therapeutic agents such as [177Lu]Lu-FAPI. However, future prospective research on a wider population is needed to confirm the excellent performance.


Introduction
Positron emission tomography/computed tomography (PET/CT) is a nuclear medicine imaging technique increasingly used in oncology for early diagnosis and treatment evaluation in several neoplasms, especially in the absence of morphological abnormalities.Although the glucose metabolism-directed fluorine-18 fluorodeoxyglucose ([ 18 F]FDG) is the mainstay of PET/CT in most applications, the availability of new, more target-specific radiopharmaceuticals evaluating different metabolic pathways and/or receptor expression has broadened the possible clinical horizons of PET/CT [1,2].Some neoplasms (e.g., prostate, renal cell carcinoma, hepatocarcinoma, or G1 neuroendocrine tumors) are not [ 18 F]FDG-avid; moreover, neoplasms growing in sites with physiologically high [ 18 F]FDG uptake (brain, oro-pharyngeal mucosa, liver, bowel, and urinary tract) may be missed.
The demonstration of molecular crosstalk between CAFs and malignant cells, leading to the secretion of growth factors and cytokines (SDF-1, HGF, FGF, IL-6, TGF-β, EGF) that promote proliferation and neo-angiogenesis [17][18][19], is the basis for using PET/CT with FAPI radiopharmaceuticals in liver malignancies.Recent clinical studies on diagnostic and/or theranostic applications of FAPI radiopharmaceuticals in oncology mostly include patients with mixed malignancies, in heterogeneous clinical settings; in patients with suspected malignant liver disease, either primary like hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), or liver metastases from various cancers, there is some evidence regarding the high diagnostic performance of FAPI radiopharmaceuticals, with it being comparable to MRI and better than [ 18 F]FDG, especially in the early stages [20][21][22].Furthermore, FAPI radiopharmaceuticals may outperform [ 18 F]FDG in detecting extrahepatic involvement from HCC or ICC [22], and they are able to differentiate ICC (with a higher TBR) from HCC and liver metastases (with a lower TBR), despite them having a limited role in predicting HCC grade [21].In patients with extrahepatic malignancies, there is initial evidence of a higher value of FAPI radiopharmaceuticals than [ 18 F]FDG in detecting liver metastases, due to a higher uptake and/or TBR [23,24].
However, to the best of our knowledge, a review exclusively focused on the diagnostic role of PET using FAPI radiopharmaceuticals in patients with primary and/or metastatic liver lesions is still lacking.Therefore, we aimed to review the available literature on this topic to provide a more comprehensive insight into the role of PET using FAPI radiopharmaceuticals in the management of patients with liver lesions.
The time interval from the tracer injection to the PET/CT image acquisition was usually 60 min, with a field of view from the vertex to the upper or mid-thighs.The injected activity was heterogeneous, from 1.8 to 3.7 MBq/Kg.In two studies performing dynamic acquisition, the scan duration ranged from 20 to 60 min [84,88].In one study, dual timepoint acquisition (at 20 and 60 min post-injection) was performed [55].
When semi-quantitative analysis was performed, maximum and mean Standardized Uptake Values (SUVmax and SUVmean) were used, as well as tumor-to-background ratio (TBR), with the normal background liver selected as a reference.
Comparison with another radiopharmaceutical was performed in 19 studies ([ 18 F]FDG in all cases); details are reported in Table 3.  Higher radiopharmaceutical uptake was mostly observed in patients with CC than in those with HCC: particularly, in 10 patients with treatment-naïve liver lesions, the [ 68 Ga]Ga-FAPI-46-derived SUVmax and TBR were significantly higher in the 5 with CC than in the 5 with HCC (24.02 vs. 7.54 and 21.07 vs. 4.98, respectively; p < 0.005) [62].Similarly, in a prospective assessment of 20 cases with liver lesions suspected of malignancy, [ 68 Ga]Ga-FAPI-04-derived SUVmax and TBRmax values were higher in ICC than in HCC (14.14 ± 2.20 vs. 8.47 ± 4.06 for SUVmax; 26.46 ± 4.94 vs. 7.13 ± 5.52 for TBRmax; p < 0.05); in the same paper, immunohistochemical analysis of samples from 7 patients with ICC demonstrated very high FAPI expression by stromal cells [20].In a prospective pilot study including 24 patients with HCC or CC, the median FAPI SUVmax and TBR for primary CC were higher than for HCC as well (CC median SUVmax 17.7 and TBR 7.14, vs. HCCs median SUVmax 11.47 and TBR 2.15) [81].Furthermore, the combination of higher uptake in lesions and lower physiological background activity in liver parenchyma resulted in a better delineation of liver lesions with [ 68 Ga]Ga-FAPI tracers compared to [ 18 F]FDG [62,87,91]: [ 68 Ga]Ga-FAPI-46 was much more sensitive than [ 18 F]FDG in both CC (100% vs. 50%) and HCC patients (100% vs. 71%) [62], and with a higher detection rate (100% vs. 57% in ICC and 94% vs. 69% in HCC) [22].
In most studies, imaging was obtained 60 min after FAPI tracer injection; in three studies, PET/CT was performed between 30 and 60 min after [ 68 Ga]Ga-FAPI administration [37,45,56].In one study, [ 68 Ga]Ga-FAPI-46 uptake was compared between 20 and 60 min post-injection acquisitions in 33 patients with suspected recurrences of pancreatic ductal adenocarcinoma, and similar detection rates were found at both timepoints in seven liver metastases (100%), along with higher SUVmax values than in reactive and cholestatic liver lesions and relatively stable TBR values across timepoints [55].[ 68 Ga]Ga-FAPI uptake was variable among studies, with median SUVmax values ranging from 2.1 to 22.2.Two studies, by Zhang et al. [27] and Qin et al. [37], used PET/MRI for image acquisition.Dynamic PET/CT images lasting 60 min were obtained in two studies: Geist BK et al. [85] performed dynamic images of the livers in 8 patients; Xing et al. [88] performed list-mode scans in 10 patients and sequential whole-body scans at 10 min intervals from 10 to 60 min post-injection in 12 patients (1 of them with liver metastases from sigmoid cancer).Moreover, Hu K et al. [84] performed a whole-body dynamic PET/CT assessment lasting 20 min in four patients with various cancers, for biodistribution assessment.
Finally, four studies [31,42,51,55] used FAP-specific radiopharmaceuticals for liver metastases without comparing them against any other tracers.Specifically, Dendl K et al. [31] demonstrated a high [ 68 Ga]Ga-FAPI uptake in liver metastases (SUVmax 9.8), comparable to that of peritoneal metastases, and an excellent TBR (8.74) with respect to the surrounding healthy parenchyma.Koerber SA et al. [42] found a high [ 68 Ga]Ga-FAPI uptake in 14 liver metastases from low gastrointestinal tract tumors (SUVmax 9.54 ± 3.74; SUVmean 4.86 ± 2.14); in these patients, the [ 68 Ga]Ga-FAPI-derived SUVmax and SU-Vmean in liver metastases were higher than in other distant sites, having been exceeded only by the primary tumor.Hoppner et al. [55] found a high TBR for liver lesions at both 20 and 60 min post-injection acquisition timepoints, although lesions were slightly more discernible in the 60 min scan due to reduced background activity.
When dynamic PET was performed, a variable uptake over time in healthy organs and liver lesions was observed.Particularly, Xing et al. [88] showed a [ 68 Ga]Ga-FAPI uptake decrease in healthy organs in the first 30 min, which occurred more slowly 30-60 min post-injection: in both protocols, the liver lesion SUVmax reached its highest value 10 min after injection (8.78 ± 3.30 in protocol 1; 12.69 ± 13.05 in protocol 2), then it was stable until 30 min post-injection (7.60 ± 4.00 in protocol 1; 11.05 ± 4.59 in protocol 2); finally, it decreased slowly from 30 to 60 min (only in protocol 2); similarly, the TBR increased from 10 to 30 min and was relatively stable in the subsequent 30 min.Furthermore, PET frames acquired at 30 and 60 min post-injection allowed researchers to detect all 56 liver lesions (12 were missed at the 10 min post-injection scan), therefore suggesting that it is appropriate to perform scans 30-60 min post-injection.However, as demonstrated by Geist et al. [85], a dynamic PET scan lasting 60 min of the upper abdomen was not able to distinguish among various malignant entities (although they showed a higher uptake than the surrounding tissue), nor was it able to distinguish inflammatory disease from normal liver parenchyma, but only to discriminate between malignant and inflammatory lesions.

FAPI PET False Positive and False Negative Findings
Concerning the interpretation of FAPI PET/CT images, attention should be paid to false positive or negative findings.Although FAP is expressed in malignant lesions' CAFs, some benign conditions exhibit an increased uptake as well [54,65,74,75,[96][97][98][99], leading to possible false positives.Non-neoplastic conditions such as inflammation after wound healing may activate the biologically quiescent fibroblasts to support immune cells and tissue cell proliferation, as part of the damage repair mechanism [100][101][102]; such activation is self-limiting once the damaging event has ceased.However, tumors may induce a persistent fibroblast activation by mimicking inflammatory cells, as "wounds that do not heal" [102,103]: in this process, CAFs contribute to tumor growth by producing pro-inflammatory cytokines and chemokines [3] that support tumor cells' proliferation, migration, invasion, angiogenesis, and resistance to therapies [3,104].
False negative findings are more frequently observed in patients with liver cirrhosis, which causes diffuse increased FAPI background liver uptake.In a patient with poorly differentiated HCC and liver cirrhosis, the uptake of [ 68 Ga]Ga-DOTA-FAPI-04 was only mild in the lesion (SUVmax 2.3) and very intense in the surrounding liver parenchyma (SUVmax 11.1) [70].Moreover, two false negative (but [ 18 F]FDG-positive) bone metastases are described in a patient with HCC [62], possibly due to FAP expression heterogeneity.

Discussion
The in-human use of radiolabeled FAPI compounds in primary and metastatic liver lesions has been rapidly growing over time, especially thanks to the usually physiological low background uptake in liver parenchyma, which makes lesion detection easier.
The available literature demonstrated the excellent diagnostic performance of FAPI PET/CT in the most common primary liver malignancies, like HCC and ICC: particularly, the high lesion uptake coupled with lower liver background uptake resulted in a higher sensitivity when compared with more "conventional" radiopharmaceuticals like [ 18 F]FDG.The TBR, which is affected by low background uptake, is the most strikingly helpful among PET semi-quantitative parameters in recognizing liver lesions [22,62].Additional intrahepatic lesions may be detected using FAPI PET/CT, leading to a more accurate staging, a better definition of prognosis, and guidance of therapeutic management [22].FAPI uptake is determined by CAFs expression in the tumor stroma: this explains the overall higher uptake in ICC than in HCC (as confirmed by CAFs' prevalence over malignant cells in the histological specimens) [22,62].Moreover, when considering histological degree in HCC patients, a lower uptake was observed in well-differentiated malignancies than in poorly differentiated ones [20], and in hematoxylin and eosin stains, it was demonstrated that more mesenchymal cells are present in less-differentiated lesions [22].A higher proportion of tumor-associated fibroblasts is a possible explanation for the intense [ 68 Ga]Ga-FAPI uptake noticed in fibrolamellar HCC [72].Recent studies have reported a significantly lower physiological liver background for [ 68 Ga]Ga-FAPI than for [ 18 F]FDG PET/CT [5,34,78,93]: particularly, Giesel et al. [34] found a normal liver mean SUVmax of 1.42 using [ 68 Ga]Ga-FAPI and of 3.10 using [ 18 F]FDG with 71 oncological patients.However, an increase in FAPI uptake may be recognized in the setting of liver cirrhosis, in terms of SUV and TBR [22,67]: this is explained by the stimulation of fibroblast activation, leading to the formation of fibrotic scars [105,106].Hepatitis may also contribute to a higher FAPI liver background uptake, especially in benign regenerative nodules; this could impact the correct detection of malignant lesions [22].The detection of cirrhosis is important in liver cancer management since it limits the spectrum of possible therapeutic strategies [22,52]; nonetheless, smallsized lesions may be missed where there is a high FAPI background uptake in cirrhotic patients [39,65,67]; we should also consider that small-sized lesions may show an absolutely lower uptake than larger-sized ones (lower hypoxia, which translates into lower FAP expression in CAFs) [67].Furthermore, FAPI PET/CT exhibited a higher detection rate than [ 18 F]FDG PET/CT, due to higher uptake in lesions and a higher TBR, making it possible to use FAPI as a promising agent for hepatic tumor staging [22].The higher expression of CAFs in the tumor stroma at early disease stages in well-and moderately differentiated lesions involves a high sensitivity of FAPI, in a clinical setting usually characterized by low [ 18 F]FDG intra-cell trapping due to enhanced glucose-6-phosphatase activity [39,67].Moreover, other less common primary liver malignancies exhibiting low [ 18 F]FDG uptake, such as neuroendocrine liver carcinoma, marginal MALT lymphoma, or PEComa, or even less common borderline primary liver lesions such as inflammatory myofibroblastoma, may benefit from the use of FAPI PET/CT, especially in patients exhibiting doubtful lesions with conventional imaging [64,66,73,76,77]. 68Ga-labeled FAPI radiopharmaceuticals have demonstrated excellent performances in identifying liver metastases from various gastrointestinal cancers: particularly, in the vast majority of the available literature, [ 68 Ga]Ga-FAPI has proven to find more liver lesions, and with higher SUVmax values, compared with [ 18 F]FDG [23,26,37,38,41,43]; moreover, [ 68 Ga]Ga-FAPI's sensitivity to liver metastases is higher than that of [ 18 F]FDG in this setting [23].However, in patients with pancreatic cancer, [ 68 Ga]Ga-FAPI PET/MR detected a lower number of liver metastases than [ 18 F]FDG, and with a lower uptake intensity [27].The TBR, as the ratio between the maximum or mean uptake of the lesion and that of background (usually physiological liver) activity, may be 2-3 times higher for [ 68 Ga]Ga-FAPI PET/CT than for [ 18 F]FDG in liver metastases from colorectal cancer (although some overlap was found) [43,45].Even in liver metastases from lung cancer, [ 68 Ga]Ga-FAPI PET/CT performs better than [ 18 F]FDG in terms of TBR (higher), despite similar SUVmax values [25,29,48].Concerning liver metastases from NETs, [ 68 Ga]Ga-FAPI detects more lesions than [ 18 F]FDG [35,39] because of the lower liver background activity; more controversial is the comparison between [ 68 Ga]Ga-FAPI and [ 68 Ga]Ga DOTATATE [35,40,56], because of a reported lower detection rate in liver metastases from NETs when using [ 68 Ga]Ga-FAPI [56].Moreover, in patients with pancreatic and intestinal NETs, FAPI PET/CT has been demonstrated to be a potentially valuable predictor of lesion aggressiveness and risk of progression, considering the very good correlation between FAPI PET/CT-derived tumor fractions and Ki-67, which is much better than the [ 18 F]FDG-derived one [24].FAPI radiopharmaceuticals perform better than [ 18 F]FDG ones, showing significantly higher TBRs and, in some cases, also more liver lesions otherwise not evident with [ 18 F]FDG, in breast cancer [36], GISTs [44,47], a case of MALT lymphoma [30], chromophobe renal cell carcinoma [46], and a case of MEN-2A syndrome [28].In a woman with medullary thyroid carcinoma, Kuyumcu S et al. [33] reported focal liver metastases only using [ 68 Ga]Ga-FAPI-04, whereas [ 68 Ga]Ga-DOTATATE showed only subtle inhomogeneity in the liver parenchyma uptake.Conversely, in some cases, such as in a reported uveal malignant melanoma [32], FAPI radiopharmaceutical uptake in liver metastases may be lower than that of [ 18 F]FDG (even lower than in an incidental inflammatory condition such as knee osteoarthritis).Two papers [14,42] have explored the diagnostic performance of 68 Ga-labeled FAP-directed radiopharmaceuticals in detecting liver metastases, not compared to other tracers: in these studies, a high [ 68 Ga]Ga-FAPI uptake was evident in liver metastases from head-and-neck, gastrointestinal, biliary-pancreatic, urinary tract, and neuroendocrine tumors, with high TBRs with respect to the surrounding healthy parenchyma [14,42], and liver metastases derived from low gastrointestinal tract tumors exhibited the highest [ 68 Ga]Ga-FAPI-04 or [ 68 Ga]Ga-FAPI-46 uptake among metastatic sites (exceeded only by the primary tumors) [42].
Dynamic acquisition has been demonstrated to be useful in depicting liver metastases, although proper acquisition timing has to be addressed for better delineation of lesions with respect to the surrounding parenchyma: the available studies [85,88] found that most lesions are most visible 30-60 min after radiopharmaceutical administration, a timing that ensures the highest TBR and an adequate lesion SUVmax (on the other hand, only a few lesions are visible in the first 10 min).However, despite FAPI PET/CT SUVmax values attained during dynamic acquisition being overall higher in malignant lesions than in normal liver parenchyma or in benign lesions, FAPI kinetics are not able to distinguish among different histologies of primary liver lesions (HCC vs. non-HCC) or between liver metastases and primary lesions, nor benign lesions from normal parenchyma.
There is recent evidence of the usefulness and safety of FAPI-directed beta-emitting compounds such as [ 177 Lu]Lu-FAPI in the treatment of liver metastases from various histologies, using FAPI PET/CT for suitability assessment and post-therapy evaluation.Barashki et al. [28] described a patient with MEN type 2A and multi-site metastases (including liver, bone, and lymph nodes) who underwent [ 177 Lu]Lu-FAPI-46 therapy on [ 68 Ga]Ga-FAPI-46 positive lesions on PET/CT, resulting in clinical improvement (resolution of abdominal pain).Baum et al. [12] have performed the first in-human study on peptidetargeted radionuclide therapy using 177 Lu-FAP-2286 to treat 68 Ga-FAP-2286-or [ 68 Ga]Ga-FAPI-04-positive liver metastases from breast, pancreas, rectum, or ovary adenocarcinomas, reporting high 177 Lu-FAP-2286 uptake in tumor lesions up to 10 days after treatment (3.0 ± 2.7 Gy/GBq), yet no Grade 4 adverse events (most patients complained of selflimiting headaches, temporary flare-up of the abdominal pain, anemia, or pancytopenia).
The first limitation to be considered for this review is the small cohort size in most included studies: indeed, although patient recruitment designs were mostly prospective, 47 out of the 76 papers included fewer than 15 patients, while 19 studies included a larger population with liver malignancies [20][21][22][23]58,61,62,67,78,[80][81][82]86,[88][89][90][93][94][95].Therefore, future trials/studies on a larger population are needed.Heterogeneity in clinical and technical aspects was also observed: most studies included a mixed population, with primary liver lesions of different histologies (HCC, ICC, or less common ones), or with liver metastases from different primary cancers (mostly gastrointestinal); moreover, different FAPI-directed radiopharmaceuticals (either 68Ga-or 18F-labeled), among studies or in the same paper, possibly with slight differences in pharmacokinetics or pharmacodynamics (affecting biodistribution in healthy organs and malignant lesions, preferred acquisition timing for best detection rate, and effective dose), were employed.Particularly, [ 68 Ga]Ga-FAPI-02 is selectively accumulated in FAP-expressing tissue, with a significantly higher uptake than that of [ 18 F]FDG in malignant lesions; [ 68 Ga]Ga-FAPI-04 is rapidly internalized into FAP-positive tumors, showing fast clearance and a very rapid accumulation in tumor sites (10 min after administration) (tumor uptake is even higher and more rapid using [ 68 Ga]Ga-FAPI-46 compared to [ 68 Ga]Ga-FAPI-04); [ 68 Ga]Ga-DOTA.SA.FAPI shows an advantage in brain metastasis compared with [ 18 F]FDG; [ 18 F]F-NOTA-FAPI-04 exhibits no significant defluorination during image acquisition; therefore, it shows a higher uptake and TBR in liver and bone tumors compared with [ 68 Ga]Ga-FAPI.Moreover, dynamic PET scans were not performed using unique protocols, and in some studies, PET/MRI was used in addition or in place of PET/CT.

Literature Search and Information Sources
A comprehensive computer literature search of the PubMed and MEDLINE databases was conducted to find relevant published articles on the diagnostic use of PET with radiolabeled FAPIs in patients with neoplastic liver lesions, including both primary liver neoplasms (such as hepatocellular carcinoma and cholangiocarcinoma) and liver metastases from any other primary malignancy.A search algorithm based on the combination of the terms ("FAPI") AND ("hepatic" OR "liver") was used.The last update of the literature search was 1st January 2024.Titles and abstracts of the retrieved studies were screened independently by two researchers (FM and GP).

Inclusion and Exclusion Criteria and Data Analysis
Only articles written in the English language and studies conducted on human subjects, that included patients with neoplastic liver lesions (primary hepatic tumors and/or liver metastases), and with available data on the diagnostic performance of PET imaging with radiolabeled FAPIs (regardless of disease stage and clinical indication for PET examination) were considered.After screening titles and abstracts, an initial selection was performed, excluding (a) articles not within the field of interest of this review (e.g., not evaluating patients with neoplastic liver lesions; with no data on PET imaging; focused on FAPI synthesis, biodistribution, pharmacokinetics, or dosimetry, or only on radiolabeled FAPI therapy); (b) pre-clinical studies; (c) review articles and meta-analyses, editorials, letters, commentaries, and conference proceedings.Then, two researchers (FM and GP) independently reviewed the full-text versions of the selected articles to assess them for final eligibility, and possible disagreements were resolved by consensus.For each included article, information was collected about the basic clinical characteristics (study design, number of patients with neoplastic liver lesions, primary liver tumors, or liver metastases, etc.), and methodological aspects (FAPI tracer, injected activity, scan delay as the interval between injection and PET image acquisition, comparison with PET imaging using other radiopharmaceuticals when available, etc.).

Conclusions
From the present review, it has emerged that the use of radiolabeled FAPI PET/CT for the study of primary and/or metastatic liver lesions, despite the evident heterogeneity in population size and in study design of the included articles, as well as in the radiopharmaceuticals employed and in histology among primary and metastatic liver lesions, carries excellent diagnostic performance, usually better than "conventional" radiopharmaceuticals: the significantly lower FAPI liver background uptake resulted in a higher sensitivity of this

Figure 1 .
Figure 1.Algorithm for article retrieval and selection.

Table 1 .
Basic, clinical, and methodological characteristics of 23 articles on primary liver tumors.

Table 2 .
Basic, clinical, and methodological characteristics of 39 articles on liver metastases.

Table 3 .
Comparison of FAPI vs. other tracer(s) in 19 studies on primary liver tumors.

Table 4 .
Comparison of FAPI vs. other tracer(s) in 35 studies on liver metastases.